CERTIFICATIONAOAC Research Institute
Performance Tested MethodsSM
Certificate No.

112201The AOAC Research Institute hereby certifies the method known as:

LuciPac A3 Surface (Pre-Moistened)
manufactured byKikkoman Biochemifa Company
2-1-1, Nishi-shinbashi
Minato-ku, Tokyo 105-0003
Japan
This method has been evaluated and certified according to the policies and procedures of the AOAC Performance Tested MethodsSM Program. This certificate indicates an
AOAC Research Institute Certification Mark License Agreement has been executed which authorizes the manufacturer to display the AOAC Research Institute
Performance Tested Methods SM certification mark on the above-mentioned method for the period below. Renewal may be granted by the Expiration Date under therules stated in the licensing agreement.

Bradley A. Stawick, Senior Director
Signature for AOAC Research InstituteIssue Date
Expiration Date

2275 Research Blvd., Ste. 300, Rockville, Maryland, USA Telephone: +1-301-924-7077 Fax: +1-301-924-7089
Internet e-mail: aoacri@aoac.org * World Wide Web Site: http://www.aoac.org

June 03, 2024
December 31, 2024

Kikkoman LuciPac A3 Surface (Pre-Moistened), AOAC Performance Tested MethodsSM Certification Number 112201

METHOD NAME
LuciPac A3 Surface (Pre-Moistened)

CATALOG NUMBER
60367

ORIGINAL CERTIFICATION DATE
November 07, 2022

PRINCIPLE OF THE METHOD
The principle of detection of A3 is shown in Figure 1 (1). Firefly luciferase can produce light in the presence of ATP, luciferin, oxygen and Mg2+. The amount of light produced is
proportional to the amount of ATP in a sample and therefore ATP can be quantified by measuring the light produced through this reaction using a luminometer, showing areading of Relative Light Units (RLUs). This is well known as the ATP method. In order to detect AMP simultaneously and maintain the light production, ATP is regenerated fromAMP using pyruvate orthophosphate dikinase reaction (PPDK) in the presence of phosphoenol pyruvate, inorganic pyrophosphate (PPi) and Mg2+ (Figure 1). Furthermore, ADP isconverted to ATP by pyruvate kinase (PK, Figure 1). This allows the test to detect and quantify total adenylate and dramatically increases the signal available to the test. BothLumitester PD-30 and Lumitester Smart detect the light with a photodiode.
D-luciferin

Oxyluciferin + CO2 + LightLuciferase
Phosphoenol pyruvate

ADPPK
Mg2+, K+Pyruvate

Mg2+

ATPAMP+ PPi

Mg2+

PPDK
Pyruvate + Pi

Phosphoenol pyruvate

Figure 1. The principle of ATP+ADP+AMP (A3) detection. Adenosine triphosphate (ATP) can be quantified by the detection of the light from the firefly luciferase reaction.
Adenosine monophosphate (AMP) is recycled to ATP using the pyruvate orthophosphate dikinase (PPDK). Adenosine diphosphate (ADP) is converted to ATP by the pyruvatekinase reaction (PK). PPi : inorganic pyrophosphate, Pi : inorganic phosphate.

Page 2 of 4Kikkoman LuciPac A3 Surface (Pre-Moistened), AOAC Performance Tested MethodsSM Certification Number 112201

CERTIFIED CLAIM STATEMENT: The LuciPac A3 Surface (Pre-Moistened) method is certified for the determination of ATP+ADP+AMP within the scope of Tables 1 and 2.
Certification includes:
1. Lumitester Smart (Cat. No. 61234) or Lumitester PD-30 (Cat. No. 60486) reader with optional temperature compensation mode.
2. Lumitester Control Kit (Cat. No. 60484) for calibration of reader.
Table 1. Method Performance Claims
SurfaceNoneAnalyte or MaterialATPATPaADPADPaAMPAMPa
Stainless SteelRaw chicken breast(10 cm x 10 cm)
Sliced deli ham
Sliced deli hama
Orange juice
Orange juicea
YogurtApple pie
Cronobacter sakazakii
Lactobacillus acidophilus
Saccharomyces cerevesiae
aTested by the Independent Laboratory.
bdf = dilution factor; cfu = colony forming unit.
c
N/A = Not applicable.

Table 2. Method Selectivity
Compounds
10 Non-target adenylates

aConcentration
2500 fmol

3 Disinfectants
ResidualInterference defined as an effect ≥20%.

Range testedb
1–100 fmol/assay
1–100 fmol/assay
1–100 fmol/assay
1–100 fmol/assay
1–100 fmol/assay
1–100 fmol/assay
1000–10,000 df
70,000–800,000 df
70,000–1,600,000 df
4000–64,000 df
4000–100,000 df
1200–12,000 df
1000–8000 df
1.8–8.5 x 104 cfu
0.6–12 x 103 cfu
40–1450 cfu

Performance Supporting Certification
Mean RLU range
LOD, fmol/assay
RSDr, %
8.6–216
7.0
6–61
11.8–212
3.1
11–23
8.5–219
4.3
6–43
13.5–218
3.7
7–30
7.7–239
5.2
6–59
11.1–235
4.9
7–22
c
61–811
N/A
6–17
37–711
N/A
21–44
39–842
N/A
8–21
41–619N/A
27–39
22–743N/A
17–26
41–808N/A
17–25
71–502
N/A
20–52
72–510N/A
6–13
60–750N/A
9–10
49–822
N/A
6–26

No. Compounds Interferinga
0 fmol ATP, ADP, AMP
100 fmol ATP, ADP, AMP
001000 fmol ATP
4000 fmol ATP
00Page 3 of 4

Kikkoman LuciPac A3 Surface (Pre-Moistened), AOAC Performance Tested MethodsSM Certification Number 112201

Table 3. Method History
No. Date1
November 2022
2May 2024

Summary
Level 2 Modification: PTM 051901 modified with a pre-moistenedswab and liquid reagents for a new certification PTM 112201.
Level 3 Modification: Modified luciferase clone; optimizedreagents; new pouch material.

Supporting Data
Certification Report (link pending)
Modification Report 1(link pending)Page 4 of 4